Pin1 Promotes Regulated Necrosis Induced by Glutamate in Rat Retinal Neurons via CAST/Calpain2 Pathway

Abstract

The purpose of the current study was to investigate whether peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) can interact with calpastatin (CAST) and regulate CAST/calpain2, under excessive glutamate conditions, and subsequently regulate necrosis in rat retinal neurons. Glutamate triggered CAST/calpain2-mediated necrosis regulation in primary cultured retinal neurons, as demonstrated by propidium iodide-staining and lactate dehydrogenase assay. Co-IP results and a computer simulation suggested that Pin1 could bind to CAST. Western blot, real-time quantitative polymerase chain reaction, immunofluorescence, and phosphorylation analysis results demonstrated that CAST was regulated by Pin1, as proven by the application of juglone (i.e., a Pin1 specific inhibitor). The retinal ganglion cell 5 cell line, combined with siRNA approach and flow cytometry, was then used to verify the regulatory pathway of Pin1 in CAST/calpain2-modulated neuronal necrosis that was induced by glutamate. Finally, in vivo studies further confirmed the role of Pin1 in CAST/calpain2-modulated necrosis following glutamate excitation, in the rat retinal ganglion cell and inner nuclear layers. In addition, a flash electroretinogram study provided evidence for the recovery of impaired visual function, which was induced by glutamate, with juglone treatment. Our work aims to investigate the involvement of the Pin1-CAST/calpain2 pathway in glutamate-mediated excitotoxicity.

Pin1 Is Regulated by CaMKII Activation in Glutamate-Induced Retinal Neuronal Regulated Necrosis

“…The morphological features of RN are similar to necrosis, which exhibit plasma integrity loss and organelle swelling, and are different from apoptosis and autophagy (Galluzzi et al, 2014; Catalani et al, 2016). Both our studies and others have found that peptidyl-prolyl isomerase 1 (Pin1) activation was involved in glutamate-induced RN (Del Rosario et al, 2015; Wang et al, 2018a). Pin1 is one subtype of the peptidyl cis- to- trans isomerases (PPIases) that can bind and catalyze cis/trans isomerization of phosphorylated threonine/serine-proline, leading to protein phosphorylation and degradation, neuronal survival and death (Agostoni et al, 2016; Islam et al, 2018).…”

“…All experimental procedures used in the present study were approved by the Ethics Committee of the 3rd Xiangya Hospital of Central South University in keeping with the Guidelines for the Care and Use of Laboratory Animals (U.S. National Institutes of Health). Primary cultures of retinal neurons were prepared from 1-day-old Sprague-Dawley (SD) rat pups as described previously (Li N. et al, 2016; Wang et al, 2017, 2018a). In brief, the eyes were removed under sterile conditions, and the retinae were extracted with the aid of a dissecting microscope.…”

“…Neuronal responses to glutamate treatment are characterized by increased intracellular calcium concentration through GluRs activation (Madji Hounoum et al, 2017). We have investigated the key molecular pathway involved in glutamate-induced RN in our previous study (Wang et al, 2018a, 2019). However, the specific role of distinct GluRs involved in the glutamate-induced RN in the retinal neuronal cultures has not been clarified.…”

Systematic spatiotemporal mapping reveals divergent cell death pathways in three mouse models of hereditary retinal degeneration

“…Prominent candidates for Ca 2 + -dependent downstream pathways are apoptotic cell death through caspase effectors (Orrenius et al, 2003) and nonapoptotic cell death involving calpain activation (Arango-Gonzalez et al, 2014;Doonan et al, 2005). To distinguish between these two proposed pathways, we used antibodies for cleaved caspase-3, a marker protein for apoptosis (Mazumder, Plesca, & Almasan, 2008), and AIF, a marker for programmed necrosis (Arango-Gonzalez et al, 2014;Wang et al, 2018).…”

Systematic spatio-temporal mapping reveals divergent cell death pathways in three mouse models of hereditary retinal degeneration

“…Prominent candidates for Ca 2+ -dependent downstream pathways are apoptotic cell death through caspase effectors (13) and non-apoptotic cell death involving calpain activation (10,14). To distinguish between these two proposed pathways, we used antibodies for cleaved caspase-3, a marker protein for apoptosis (42), and AIF, a marker for programmed necrosis (10,43).…”